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Abstract

Background

Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels
are associated with impaired endothelial function in type 2 diabetes (T2D). In this
work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production
by endothelial cells in vitro.

Methods

In human umbilical vascular endothelial cells (HUVECs), we measured the effects of
FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression
and activation and on NO production. We also explored the impact of exogenous FABP4
on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt).

Results

We found that eNOS expression and activation and NO production are significantly inhibited
by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated
eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4
induces endothelial dysfunction by inhibiting the activation of the insulin-signalling
pathway resulting in decreased eNOS activation and NO production.

Conclusion

These findings provide a mechanistic linkage between FABP4 and impaired endothelial
function in diabetes, which leads to an increased cardiovascular risk.

Keywords:

Introduction

The adipose fatty acid-binding protein (FABP), also known as FABP4 and aP2, is one
of the best characterised intracellular lipid transport proteins [1]. It belongs to the superfamily of low-molecular-weight intracellular lipid-binding
proteins and plays a central regulatory role in energy metabolism and inflammation
[2,3]. FABP4 is highly expressed in mature adipocytes and accounts for approximately 6 %
of their soluble protein. FABP4 is also found circulating in the plasma. In the last
several years, much effort has been focused on uncovering the role of FABP4. However,
neither the secretory pathways nor the functions of circulating FABP4 are known. We
and other authors have shown that FABP4 levels are increased in obesity, metabolic
syndrome (MS), type 2 diabetes (T2D), and familial combined hyperlipidaemia or lipodystrophy
syndromes, and these levels are also closely correlated with adverse lipid profiles
and insulin resistance [4-10]. In these studies, serum FABP4 predicted the development of MS and atherosclerosis
[11,12].

A recent study showed that although serum levels of both adipocyte and epidermal FABP
had associations with MS, only FABP4 was significantly associated with increased cardiovascular
risk in Chinese adults [13]. Moreover, increased plasma levels of FABP4 in non-elderly men were independently
associated with the presence of coronary artery disease [14]. A recent study showed that FABP4 had a direct impact on decreasing the contractility
of myocardial muscle cells, which suggested that the release of FABP4 in to the bloodstream
could have a direct effect on some peripheral cells and tissues [15]. In addition, we recently demonstrated that high levels of plasma FABP4, as other
inflammation mediators, were associated with endothelial dysfunction assessed by peripheral
artery tonometry [16,17].

Endothelial dysfunction is the first event in the pathogenesis of atherosclerosis
and refers to an imbalance in the release of vasodilating molecules, such as nitric
oxide (NO) and vasoconstricting factors. NO-dependent vasodilatation is thought to
reflect endothelial function, and its impairment is predictive of future cardiovascular
risk [18,19]. The insulin-signalling pathway in the vascular endothelium leads to the activation
of endothelial nitric oxide synthase (eNOS) and an increased production of NO. This
pathway involves the insulin receptor-mediated phosphorylation of insulin receptor
substrate 1 (IRS1), which activates PI3-kinases that then phosphorylates and activates
Akt at Ser473. Akt directly phosphorylates eNOS at Ser1177, resulting in increased eNOS activation and NO production. Under pathological conditions,
proinflammatory factors cause an impairment in this particular insulin-signalling
pathway in the endothelium, which promotes endothelial dysfunction [20]. This impairment might be related to the defective insulin signalling in the endothelial
cells. Moreover, insulin may also activate the pro-atherogenic mitogen-activated protein
kinase (MAPK) pathway in endothelial cells that leads to increases in the expression
of adhesion molecules and leukocyte adhesion to the vascular endothelium. In addition,
experimental animals with a vascular endothelial cell-specific insulin receptor deficiency
show a reduction in the expression of adhesion molecules and eNOS mRNA [21].

Although the role of circulating FABP4 on the vascular endothelium is unknown, a recent
study has revealed that the elevated expression of intracellular FABP4 in endothelial
cells contributes to their dysfunction through a reduction of eNOS [22]. These data along with our own observations showing the influence of circulating
FABP4 on endothelial function warrants testing the hypothesis that high levels of
circulating FABP4 in altered metabolic conditions could modify the correct function
of endothelial cells and cause endothelial dysfunction by impairments of the insulin-signalling
pathway and NO production. If this hypothesis were correct, it would contribute to
the mechanisms by which circulating FABP4 contributes to vascular endothelial dysfunction
in diabetes.

Materials and methods

Cell culture and reagents

Human umbilical vein endothelial cells (HUVECs) were obtained from Cascade Biologics
(Invitrogen Life Technologies, UK). After thawing, cells were seeded in 75-cm2 flasks and cultured in medium 200 according to the supplier’s recommendations. Medium
200 was supplemented with 2 % low serum growth supplement (LSGS) and 1 % Gentamicin/Amphotericin
solution (Invitrogen Life Technologies, UK). The cells were placed in a humidified
incubator at 37 °C and 5 % CO2 until there were enough cells available for experiments. The HUVEC cells were used
at passage 3 in the current study.

Design of the studies to determine the FABP4 effects

Effect of FABP4 on eNOS activation

Confluent cells were starved in 1 % low serum medium (without growth factors). To
determine whether FABP4 has any effect on the basal eNOS activation, we studied eNOS
phosphorylation at Ser1177. The HUVECs were incubated with FABP4 (25–100 ng/ml) for 30 min in the endothelial
starvation medium. The treated cells were rinsed with ice-cold PBS and lysed in lysis
buffer, which was composed of 50 mM Tris–HCl, 150 mM NaCl, 0.1 % SDS, 1 % Nonidet,
0.5 % deoxycholate and protease inhibitors, and the cells were stored at −80 °C until
they were processed. The total protein concentration was measured using a Bradford
assay (BioRad, USA). Immunoblot analysis of eNOS phosphorylation at Ser1177 and total eNOS was performed.

We also examined the effect of FABP4 on the insulin-stimulated eNOS phosphorylation
at Ser1177. The HUVECs were preincubated with FABP4 (25–100 ng/ml) for 30 min and were then
stimulated with 600 nM insulin for 30 min in the endothelial starvation medium. We
used the same protocol described above to obtain the samples. The total protein concentration
was measured using a Bradford assay. Immunoblot analysis of eNOS phosphorylation at
Ser1177 and total eNOS was also performed.

Effect of FABP4 on the insulin-signalling pathway

Because insulin activates eNOS through IRS1 and Akt, we investigated whether FABP4
would have any effect on the insulin-stimulated IRS1 and Akt activation. We studied
the phosphorylation of IRS1 at Tyr989 and of Akt at Ser473. The HUVECs were preincubated for 30 min with FABP4 (25–100 ng/ml) before a 30-min
insulin treatment (600 nM) in the starvation medium. Treated cells were collected
and lysed in lysis buffer, and then the lysates were stored at −80 °C until they were
processed. The total protein concentration was measured using a Bradford assay. Immunoblot
analysis of IRS1 phosphorylation at Tyr989, total IRS1, Akt phosphorylation at Ser473 and total Akt was performed.

Immunoblot analysis of eNOS phosphorylation at Ser1177, total eNOS, IRS1 phosphorylation at Tyr989, total IRS1, Akt phosphorylation at Ser473, and total Akt, and FABP4

Electrophoresis and immunoblot analysis were performed using the NuPAGE Protein Analysis
System (Invitrogen Life Technologies, UK). The membrane was blocked with a 2 % ECL
Advance Blocking Reagent (Amersham Biosciences, Fairfield, CT) and was incubated with
anti-eNOS, anti-eNOS phosphorylation at Ser1177, anti-Akt, anti-Akt phosphorylation at Ser473, anti-IRS1 phosphorylation at Tyr989, anti-IRS1, or anti-FABP4 antibodies. Antigen-antibody complexes were detected by
incubating the membrane with an IgG-HRP antibody. The bands were visualised using
ECL reagents (Amersham Pharmacia, Fairfield, CT) with the VersaDoc image system and
were quantified with the Quantity One analysis software version 4.6.2 (Bio Rad, USA).
The relative levels of the phosphorylated proteins were quantified after being normalised
to the total proteins and were expressed as arbitrary units (AU).

Effect of FABP4 on eNOS mRNA expression

Upon insulin stimulation, the eNOS mRNA was increased in the vascular endothelial
cells. Therefore, we examined the mRNA expression of insulin-stimulated eNOS from
the FABP4-treated cell lysates. Confluent vascular cells were preincubated with FABP4
(50–100 ng/ml) for 30 min and then stimulated with 600 nM insulin for 24 hours in
a supplemented medium. Total RNA was isolated from the cells using the ABI PRISM 6100
Nucleic Acid PrepStation (Applied Biosystems, CA, USA). The absorbance at 260 nm was
used to measure the RNA concentration, and an absorbance ratio of 260/280 nm was used
to analyse the RNA quality.

Real-time quantitative PCR of eNOS

Total RNA (0.5 μg) was reverse transcribed to cDNA using Random Hexamers and SuperScript
II (Invitrogen Life Technologies, UK) by following the manufacturer’s protocol. TaqMan
primers and probes for eNOS and GAPDH were obtained from validated and pre-designed
Assays-on-Demand products (Applied Biosystems, CA, USA) and were used in real-time
PCR amplifications. The mRNA expression for each gene and sample was calculated using
the recommended 2-ΔΔCt method. The control group (untreated cells) was defined as the calibrator in this
experiment. GAPDH was used as a housekeeping gene to normalise the results of the
gene of interest.

Nitric oxide assay

Cells were starved for 24 h with 1 % low serum medium (without growth factors) and
then preincubated for 30-min with FABP4 (25–100 ng/ml) before a 30-min insulin treatment
(600 nM) in the starvation medium. The supernatants were collected, and the detection
of the NO2 and NO3 anions was performed with the Nitrate/Nitrite Colorimetric Assay kit (Cayman Chemical,
Ann Arbor, MI) using the Griess reaction and following manufacturer’s instructions.
The absorbance of the solution was read on a spectrophotometer at 540 nm. To quantify
the NO production, a standard nitrate curve was generated in the same medium in which
the experiments were performed. The results were expressed as fold increases with
respect to the insulin treatment.

Statistical analyses

The results are represented as the means ± SD of at least 3 separate experiments.
Differences between the means were determined using a one-way analysis of variance
(ANOVA), which was followed by a Dunnett’s post-hoc test for multiple comparisons.
Differences were considered significant at P < 0.05. The GraphPad Prism 5.0 Software, Inc. was used for statistical analyses.

Results

FABP4 inhibits eNOS activation and NO production in HUVECs

To determine whether FABP4 have any effect on eNOS activity, we studied the eNOS activation
measuring its phosphorylation at Ser1177. We found that exposing HUVECs in the endothelium growth medium (with serum and growth
factors that maintain the basal activation of eNOS) to FABP4 (25-100 ng/ml) inhibited
eNOS phosphorylation at Ser1177 in a concentration-dependent manner at as early as 30-min of exposure. The maximal
inhibition was achieved at 100 ng/ml of FABP4 (65 %; P < 0.05) (Figure 1A). On insulin stimulation, eNOS is phosphorylated by the activation of PI3k/Akt pathway
in vascular endothelial cells. First, we performed a dose–response curve with 100,
300 and 600 nM insulin, and we observed that 600 nM insulin was required to increase
eNOS Ser1177 phosphorylation by 20 % (P < 0.05) (Figure 1B). Therefore, we performed all of the experiments with 600 nM insulin. We also performed
an insulin time course (10, 30, 60 min), and we observed that the maximal phosphorylation
of eNOS at Ser1177 occurred 30 min after insulin treatment (26 % increase; P < 0.05) (Figure 1C). We next analysed whether FABP4 also impairs insulin–induced eNOS activation and
NO production. As shown in Figure 1D, the addition of insulin increases the phosphorylation of eNOS at Ser1177, but a 30-min pretreatment with FABP4 (25–100 ng/ml) inhibits this insulin-dependent
increase by up to 45 % with the 100-ng/ml concentration (P < 0.05) in the absence
of any changes in the total protein levels. Therefore, we examined the effect of FABP4
on the ability of eNOS to produce NO under insulin-stimulated conditions. The change
in eNOS phosphorylation at Ser1177 was accompanied by a significant decrease in the NO production of up to 68 % by treatment
with 100 ng/ml of FABP4 (P < 0.05) (Figure 1E). Thus, FABP4 can inhibit both the basal and the insulin-stimulated eNOS phosphorylation
at Ser1177 and can cause the inactivation of eNOS and decrease NO production in endothelial
cells.

Figure 1.Effect of FABP4 on eNOS activation and NO production in HUVECs. A, effect of FABP4 on eNOS phosphorylation at Ser1177. HUVECs were incubated with FABP4 at the indicated concentrations for 30 minutes.
B, eNOS Ser1177 phosphorylation in response to various doses of insulin (100, 300, 600 nM). HUVECs
were incubated with insulin at the indicated concentrations for 30 minutes. C, Time course of eNOS Ser1177 phosphorylation in response to insulin. HUVECs were incubated with 600 nM insulin
for 10, 30 and 60 min. D, effect on insulin-stimulated eNOS phosphorylation at Ser1177. HUVECs were incubated with FABP4 at the indicated concentrations for 30 minutes
and then with insulin (600 nM) for 30 minutes. E, effect of FABP4 on NO production. HUVECs were incubated with FABP4 at indicated
concentrations for 30 minutes and then stimulated with insulin (600 nM for 30 min).
Representative blots are shown. The data are given as the mean ± standard deviation
from three independent experiments. *P < 0.05 vs. insulin(−) / FABP4(−); #P < 0.05 vs. insulin(+) / FABP4(−).

FABP4 inhibits insulin-stimulated eNOS mRNA expression in HUVECs

Upon insulin stimulation, the eNOS mRNA was increased in the vascular endothelial
cells. As was expected, insulin augmented the mRNA expression of eNOS after 24 hours
of treatment (P <0.05). The upregulation of insulin-stimulated eNOS expression was inhibited by 63 %
and 59 % due to the treatment with 50 ng/ml and 100 ng/ml of FABP4, respectively (P < 0.05) (Figure 2). Additionally, we found a 93 % decrease in the eNOS expression after an exposure
to TNFα (10 ng/ml) as positive control (P < 0.05).

Figure 2.Effect of FABP4 on eNOS mRNA expression. HUVECs were incubated with FABP4 at the indicated concentrations for 30 minutes and
then with insulin (600 nM) for 24 hours. TNFα (10 ng/ml) was used as a negative control.
The data are expressed using the 2-ΔΔCt method. *P < 0.05 vs. insulin(−) / FABP4(−); #P < 0.05 vs. insulin(+) / FABP4(−).

FABP4 effect on eNOS is produced through the insulin-signalling pathway in HUVECs

Since insulin activates eNOS through IRS1 and Akt pathway, we then investigated whether
FABP4 have any effects on insulin-stimulated IRS1 and Akt activation, which was monitored
by phosphorylation at Tyr989 and Ser473 sites, respectively. The addition of insulin to HUVECs increases about 25 % the phosphorylation
of IRS1 at Tyr989 (Figure 3A), but a 30-min pretreatment with FABP4 (25–100 ng/ml) inhibits this insulin-dependent
increase by up to 44 % with the 100-ng/ml concentration (P < 0.05). No variations
were observed in the total IRS1 protein level across the different FABP4 concentration
treatments. There was also a similar effect of the FABP4 treatment on the phosphorylation
of Akt at Ser473. The insulin stimulation significantly increased the phosphorylation of Akt at Ser473 (Figure 3B). The FABP4 pretreatment impaired this insulin-mediated phosphorylation of Akt at
Ser473 by up to 75 % with the 100 ng/ml concentration (P < 0.05) in the absence of any change
in protein levels. Collectively, FABP4 inhibited insulin-stimulated phosphorylation
of IRS1 and Akt, indicating possible impairment of upstream insulin-signalling pathway.

Figure 3.Effect of FABP4 on IRS1 and Akt activation in HUVECs.A, effect on insulin-stimulated IRS1 phosphorylation at Tyr989. B, effect on insulin-stimulated Akt phosphorylation at Ser473. HUVECs were incubated with FABP4 at the indicated concentrations for 30 minutes
and then with insulin (600 nM) for 30 minutes. Representative blots are shown. The
data are given as the mean ± standard deviation from three independent experiments.
*P < 0.05 vs. insulin(−) / FABP4(−); #P < 0.05 vs. insulin(+) / FABP4(−).

In Figure 4, we analysed whether FABP4 was able to be internalized within the endothelial cells.
We observed that incubation of HUVECs with 25–100 ng/ml FABP4 for 30 min increased
the amount of exogenous FABP4 in the treated cells compared with the untreated cells
(P < 0.05).

Figure 4.FABP4 analysis in total cell lysates. HUVECs were treated with the indicated concentrations of FABP4 for 30 minutes followed
by 600 nM insulin for 30 minutes. Representative blots are shown. The data represent
the mean ± standard deviation from three independent experiments. *P < 0.05 vs. the untreated contro.

Discussion

The present study demonstrates that exogenous FABP4 induces endothelial cell dysfunction
in vitro, as assessed by the impact on one of their main properties, the vasodilatory mechanisms.
We have also shown that this effect is mediated by the interaction of FABP4 with the
insulin-signalling pathway in vascular cells. FABP4 alters eNOS activation, as was
demonstrated by the reduction of eNOS phosphorylation at Ser1177 and NO production. In addition, the reduction of IRS1 phosphorylation at Tyr989 and Akt phosphorylation at Ser473 suggests that the effect observed for eNOS activation is due to the interference
of FABP4 in the insulin-signalling pathway in endothelial cells. Our results support
a direct effect of extracellular FABP4 on vascular cells and, therefore, a putative
effect of circulating FABP4 on peripheral tissues. This observation is important because
FABP4 is recognised as a biomarker of cardiometabolic risk but it could be considered
as mediator of peripheral tissue damage. Several studies have linked FABP4 levels
to obesity, T2D and MS [4,8,11]. Additionally, FABP4 has been associated with the burden of coronary atheromatosis,
but a causal role has not yet been established. The only report that showed a direct
effect of FABP4 on cells demonstrated that FABP4 reduces the contractile capacity
of cardiomyocytes [15]. We have previously reported that plasma FABP4 levels are associated with endothelial
dysfunction in diabetic patients [16]. Our present data supports a causal role of FABP4 in the dysfunction of the vascular
wall. These findings are in accordance with a previous study suggesting that an elevated
expression of intracellular FABP4 in vascular endothelial cells contributes to endothelial
dysfunction both in vivo and in vitro[22]. In contrast, a recent study showed that FABP4/5 inhibitors ameliorate dyslipidaemia
but not insulin resistance in diet-induced obese mice [23]. Our results show that exogenous FABP4 alters the insulin-signalling pathway at its
early activation steps. Additionally, it has already been shown that FABP4 interferes
with the insulin receptor [24]. Moreover, FABP4 could be phosphorylated on Tyr19 in response to insulin stimulation [25]. Our results support these previous observations and extend them to the exogenous
FABP4, which has a greater clinical implication. Along with a decrease in eNOS expression,
in our hands, FABP4 increased the expression of vascular cell adhesion protein 1 (VCAM1),
E-selectin and leukocyte adhesion to endothelial cells (data not shown), suggesting
a more global effect of FABP4 on endothelial function. In our experiments, HUVECs
were stimulated with 600nM insulin because that was the dose with which we observed
an increase in the phospho-eNOS produced and because other authors also obtained maximal
nitric oxide production with similar dose [26].

The mechanisms that FABP4 utilises to interfere with the cell are currently being
investigated. Although previous studies showed that Heart-FABP (62 % homology with
FABP4) has the capability of binding to a membrane receptor described in cardiac cells
[27], this observation has neither been confirmed nor extended to other FABP family members.
It has been shown that heart FABP levels in serum could represent a useful biomarker
for myocardial function in pre-diabetic patients [28]. It is not known whether FABP4 interacts directly with the insulin receptor or if
it interacts with other components of the cell membrane and then secondarily modifies
the insulin cascade. It is also unknown whether circulating FABP4 can be internalised
into the cell to act by intracellular mechanisms. We have observed that the amount
of intracellular FABP4 increases after FABP4 incubations with respect to non-treated
cells, which suggests that FABP4 could be internalised by endothelial cells. FABP4
is an intracellular long-chain fatty acid transporter [1,29]. Although this function has not yet been demonstrated for circulating FABP4, we cannot
exclude that the effects associated with extracellular FABP4 could be mediated by
fatty acids, which are molecules known to play a role in the dysfunction of the insulin-signalling
pathway. This finding could be important evidence that FABP4 plays a pivotal role
in impairing the insulin signalling pathway.

Our findings suggest that high levels of FABP4 in the plasma are not just a clinical
manifestation of insulin resistance but also a causative factor of the development
of insulin resistance at the vascular level leading to NO metabolism alteration.

Conclusions

In summary, we have demonstrated that FABP4 can modulate the insulin-signalling pathway
in vascular cells and, consequently, decrease eNOS activation and NO production, which
impairs arterial vasodilatation. Our results supports that FABP4 plays a direct causal
role in endothelial dysfunction and could be a good therapeutic target for patients
with T2D.

Competing interests

Authors’ contribution

GA conducted the experimental work with contributions from JG, GA, PS, MH, AC, JG
and LM contributed to method development, establishment of cell lines, experimental
design and data interpretation. GA, JG and LM wrote the paper. All authors read and
approved the final paper.

Acknowledgements

This work was supported by grants from the ISCIII, Madrid, Spain (PI 05/1954, PI 10/02547,
FEDER) and from the CIBER in Diabetes and Associated Metabolic Disorders (ISCIII,
Ministerio de Ciencia e Innovación), Spain.